Approaches using genetically engineered fluorescent protein (FP) have revolutionized cell and molecular biology. Fluorescent proteins have worked as reporters within a living cell by tagging protein, allowing for the understanding of protein functions by visualizing molecular motions and localizations in real time. Meanwhile, with respect to DNA molecules, there are studies on the migration of particular proteins on the DNA molecules using fluorescent proteins, but there have been no reported study results about the staining of DNA molecules per se using fluorescent proteins in order to visualize DNA molecules.
DNA molecules are usually stained by fluorescent organic dyes. For example, intercalating dyes of ethidium bromide (EtBr) are widely used for staining DNA after gel electrophoresis. Under a fluorescent microscope, large DNA molecules are often visualized by a bis-intercalating dye of oxazole yellow homodimer (YOYO) (1). Within a living cell, DNA is often stained by membrane permeable dyes such as SYTO (2).
Such intercalating dyes have advantages of staining DNA through a simple procedure and showing high signal-to-noise ratios and low dissociation constants (Kd=12.2 μM for EtBr, Kd=12.1 nM for YOYO-1) (1, 2). However, these organic dyes have the following disadvantages due to intrinsic characteristics thereof. Since the organic dyes are ex vivo induction materials, they act as a potential mutagen, and thus the attention of researchers is required. When the organic dyes are irradiated with a laser for fluorescence, a radical intermediate is formed, causing the photocleavage of DNA phosphoric acid backbone, and thus samples cannot be further used in tests (3-7). In order to solve these problems, many researchers are endeavoring to develop safe and convenient DNA staining materials.
Numerous journal articles and patent documents are referred to herein over the entire specification and indicated as referred to. The disclosure of the cited journal articles and the patent documents are incorporated herein in their entirety by reference to further elucidate the level of the technologies to which the present invention belongs and the details of the present invention.